The role of beta-synuclein in synaptic vesicle release

项目摘要/摘要 α-突触核蛋白(α-syn)和β-突触核蛋白(β-syn)是在脊椎动物中广泛表达的蛋白质 神经系统。α-SYN聚集与帕金森氏病(PD)、多系统病理相关 萎缩症、路易体痴呆(LBD)和许多其他联体核病。已知βSYN参与了NeU- 退行性疾病，如LBD、弥漫性路易体病、高谢病和帕金森病，但几乎 人们对它的生理功能一无所知。初步数据表明，β系统直接相互作用 与α同步导致α同步的突触小泡结合减少，潜在地影响大脑中的α同步功能。 本提案的目标是描述β系统与α系统相互作用的功能后果 对于一个神经元来说。这项建议的中心假设是，基于强大的初步数据，βSYN降低了 αSYN能够支持SNARE-Complex组装、突触小泡聚集，从而支持神经元- 发射机释放。这些研究的基本原理是了解β同步对α同步功能的影响 可能导致对突触核蛋白在大脑中的功能的新的理解，并可能为以下研究奠定基础 通过调节β突触来替代α突触来对抗突触核病的新策略 直接去吧。在初步数据的指导下，这一假设将在两个具体目标中进行检验：目标1)确定 β-Syn对α-Syn介导的SNARE-Complex组装和突触囊泡聚集的影响 我的β系统是如何影响神经元活动的。在第一个目标中，圈套-复合体水平和突触小泡簇- 在P40WT和αSyn KO小鼠脑组织中，ING将被量化为不同的βSyn/βSyn比率的函数 在选定的大脑区域和具有不同βSYN水平的初级神经元中进行击打。在第二个目标中，更改为 慢病毒表达的βSyn KO神经元自发和诱发性神经递质释放 将使用微电极阵列和突触Hluorin实验来测量βSYN的折痕量。 我们期望βSYN能够减少αSYN介导的SNARE-Complex组装和突触小泡聚集。我们也 期待βSYN通过抑制αSYN而减少αSYN对突触囊泡释放和周期的影响 从突触小泡向更易溶解和功能不活跃的池转移。这项研究具有重要的意义 因为了解β系统如何发挥作用的分子机制拓宽了我们对 所有突触核蛋白的功能，并可能提供对α突触核病中突触核蛋白功能障碍的洞察。以前的工作 专注于从神经元中消除αSYN已被证明是有害的。更改β同步而不是A同步 直接治疗可能提供一种潜在的治疗途径，但仍有许多研究要做。这项研究是 创新是因为(1)它提出了β同步影响α同步功能的新假设，(2)拟议的研究直接 解决了对β突触蛋白的分子功能及其对α突触蛋白的影响缺乏了解的问题，这可能会打开 新的联合核病疗法的研究途径，以及(3)其多学科方法结合- ING生化和细胞生物学方法，以深入了解β同步蛋白的生理功能。

PROJECT SUMMARY/ABSTRACT α-Synuclein (αSyn) and β-synuclein (βSyn) are abundantly expressed proteins found throughout the vertebrate nervous system. αSyn aggregation is pathologically associated with Parkinson’s disease (PD), multiple system atrophy, Lewy body dementia (LBD), and many other synucleinopathies. βSyn is known to be involved in neu- rodegenerative diseases such as LBD, diffuse Lewy body disease, Gaucher’s disease, and PD, but almost nothing is known about its function physiologically. Preliminary data demonstrate that direct interaction of βSyn with αSyn results in reduced synaptic vesicle binding of αSyn, potentially affecting αSyn function in the brain. The objective of this proposal is to delineate the functional consequences of the interaction of βSyn with αSyn for a neuron. The central hypothesis of this proposal, based on strong preliminary data, is that βSyn reduces αSyn’s ability to support SNARE-complex assembly, synaptic vesicle clustering and thereby neuro- transmitter release. The rationale for these studies is that understanding the effects of βSyn on αSyn function may lead to a novel understanding of synuclein function in the brain, and may lie the groundwork for the devel- opment of novel therapeutic strategies for combating synucleinopathies by modulating βSyn instead of αSyn directly. Guided by preliminary data, this hypothesis will be tested in two specific aims: Aim 1) Determine the effect of βSyn on αSyn-mediated SNARE-complex assembly and synaptic vesicle clustering, and Aim 2) Deter- mine how βSyn affects neuronal activity. In the first aim, SNARE-complex levels and synaptic vesicle cluster- ing will be quantified as a function of varied αSyn/βSyn ratios, in P40 WT and βSyn KO mouse brain tissue punches of select brain regions, and in primary neurons with varied βSyn levels. In the second aim, changes to spontaneous and evoked neurotransmitter release in βSyn KO neuronal cultures lentivirally expressing in- creasing amounts of βSyn will be measured, using a microelectrode array and synaptopHluorin experiments. We expect βSyn to reduce αSyn-mediated SNARE-complex assembly and synaptic vesicle clustering. We also expect βSyn to reduce the effect of αSyn on synaptic vesicle release and cycling, due to restraining αSyn away from synaptic vesicles and towards a more soluble and functionally inactive pool. This research is significant because understanding the molecular mechanism of how βSyn functions broadens our understanding of the function of all synucleins, and may provide insight into αSyn dysfunction in synucleinopathies. Previous work focused on eliminating αSyn from neurons was has shown to be detrimental. Altering βSyn instead of aSyn directly may provide a potential therapeutic avenue, but much research remains to be done. This research is innovative because of (1) its novel hypothesis that βSyn affects αSyn function, (2) the proposed study directly addresses the lack of knowledge into the molecular function of βSyn and its effect on αSyn, which could open up avenues of research for new synucleinopathies therapeutics, and (3) its multidisciplinary approach combin- ing biochemical and cell biological approaches to gain insight into the physiological function of βSyn.